Process for providing PEGylated protein composition
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Publication Number
US-12208142-B2
Publication Date
2025-01-28
Expiration Date
Abstract
Processes for providing a PEGylated protein composition comprising mono-PEGylated protein and oligo-PEGylated protein, and processes for providing mono-PEGylated protein compositions with high yield and productivity are provided. The processes are particularly suitable for providing mono-PEGylated erythropoietin composition. The processes comprise reacting a non-PEGylated protein with a PEGylation reagent to produce a mixture comprising non-PEGylated, mono-PEGylated and oligo-PEGylated protein, subjecting the mixture to a step of anion exchange chromatography, and recycling non-PEGylated protein into further PEGylation reactions.
Core Innovation
The invention relates to producing a PEGylated protein mixture by reacting a non-PEGylated protein with a PEGylation reagent to form a mixture of reaction products comprising non-PEGylated protein and PEGylated protein. The PEGylated protein comprises mono-PEGylated protein and oligo-PEGylated protein, and the process is directed to PEGylating erythropoietin.
The process subjects the mixture of reaction products to an ion exchange chromatography (IEC) step that is an anion exchange (AEC) step. The mixture is applied to an IEC material under conditions suitable for binding non-PEGylated protein, and the IEC flow-through solution provides a PEGylated protein mixture comprising mono-PEGylated protein and oligo-PEGylated protein, with an increased fraction of PEGylated protein relative to the mixture of reaction products.
Non-PEGylated protein is recovered by eluting an IEC eluate from the IEC material and is used in a subsequent PEGylation reaction, enabling repeated production cycles using the recovered starting material. The IEC material has a binding capacity for the non-PEGylated protein of at least about 20 g/L and the PEG/protein molar ratio in the PEGylation reaction is about 0.6-1.0.
Claims Coverage
The document includes one independent claim covering a PEGylated protein mixture production process. The inventive features center on PEGylation to form mono-PEGylated and oligo-PEGylated protein, an AEC-based IEC flow-through enrichment step that binds non-PEGylated protein, and recovery of non-PEGylated protein for subsequent PEGylation reactions, with erythropoietin and quantitative constraints specified in dependent coverage.
PEGylation producing mono-pegylated and oligo-pegylated protein
Reacting a non-PEGylated protein with a PEGylation reagent to produce a mixture of reaction products comprising non-PEGylated protein and PEGylated protein, wherein the PEGylated protein comprises mono-PEGylated protein and oligo-PEGylated protein.
Anion exchange IEC flow-through enrichment
Subjecting the mixture of reaction products to an ion exchange chromatography (IEC) step to provide an IEC flow-through solution in which the fraction of PEGylated protein is increased relative to the mixture of reaction products, wherein the IEC step is an anion exchange (AEC) step and the mixture is applied to an IEC material under conditions suitable for binding non-PEGylated protein.
Recovery and reuse of non-pegylated protein
Collecting the IEC flow-through solution to provide a PEGylated protein mixture comprising mono-PEGylated protein and oligo-PEGylated protein, and recovering non-PEGylated protein by eluting an IEC eluate from the IEC material for use in a subsequent PEGylation reaction.
Erythropoietin process constraints
Wherein the protein is erythropoietin, wherein the IEC material has a binding capacity for the non-PEGylated protein of at least about 20 g/L, and wherein the PEG/protein molar ratio in the PEGylation reaction is about 0.6-1.0.
The claims cover PEGylation followed by an AEC/IEC flow-through step that enriches PEGylated protein by binding non-PEGylated protein, while recovering the non-PEGylated fraction for reuse, with erythropoietin and binding-capacity and PEG/protein ratio constraints specified in dependent coverage.
Stated Advantages
Higher yield and productivity vs prior CEC-based approaches.
Elimination of diafiltration/ultrafiltration for recycling due to low-salt IEC eluates.
Documented Applications
Producing PEGylated protein mixtures including mono-PEGylated and oligo-PEGylated forms, specifically including mono-PEGylated erythropoietin (EPO).
Purifying PEGylated protein mixtures to separate and enrich mono-PEGylated protein, including by HIC flow-through to increase the mono-PEGylated fraction.
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